This invention relates to a novel method for the chemical synthesis of oligonucleotides, including RNA, DNA, chimeric oligonucleotides, and chemically modified nucleic acids. Specifically, the invention concerns novel processes for synthesis of oligonucleotides using controlled pore glass solid support.
The following is a discussion of relevant art, none of which is admitted to be prior art to the present invention.
Chemical synthesis of oligonucleotides can be accomplished using a number of protocols, including the use of solid support chemistry, where an oligonucleotide is synthesized one nucleoside at a time while anchored to an inorganic polymer. The first nucleotide is attached to an inorganic polymer using a reactive group on the polymer, which reacts with a reactive group on the nucleoside to form a covalent linkage. Each subsequent nucleoside is then added to the first nucleoside molecule by: 1) formation of a phosphite linkage between the original nucleoside and a new nucleoside with a protecting group; 2) conversion of the phosphite linkage to a phosphate linkage by oxidation; and 3) removal of one of the protecting groups to form a new reactive site for the next nucleoside (Caruthers & Matteucci, U.S. Pat. No. 4,458,066; U.S. Pat. No. 5,153,319; U.S. Pat. No. 5,132,418; U.S. Pat. No. 4,973,679 all of which are incorporated by reference herein). Solid phase synthesis of oligonucleotides eliminates the need to isolate and purify the intermediate products after the addition of every nucleotide base.
Following the synthesis of RNA, the oligonucleotides is deprotected (Wincott et al., supra) and purified to remove by-products, incomplete synthesis products, and the like.
The demand for oligonucleotides for use as therapeutic agents, diagnostics, and research reagents has created the need for the efficient cost effective large scale manufacture of these compounds. Currently, efforts have focused on improving the coupling efficiency and the maximization of yield in phosphoramidite based synthesis. However, another area that deserved attention in this approach is the overall time and cost of preparing the nucleoside phosphoramidite reagents to be used as raw materials in the manufacture of oligonucleotides. The use of in situ phosphoramidite generation in the synthesis of oligonucleotides is an attempt to overcome the limitations imposed on the synthesis and isolation of phosphoramidites. By generating reactive nucleoside intermediates during the actual synthesis of the oligonucleotide, the need for separate phosphoramidite manufacture is overcome. However, efforts thus far have relied upon the in situ generation of 5′-O-protected nucleoside 3′-O-phosphoramidites that are coupled with 5′-OH nucleophiles. This approach is problematic in that dimerization of the intended 5′-O-protected nucleoside 3′-O-phosphoramidites occurs as a competing reaction, thereby reducing the effective equivalents of phosphoramidite available for coupling.
Tracz, U.S. Pat. No. 5,686,599, describes a method for one pot deprotection of RNA under conditions suitable for the removal of the protecting group from the 2′ hydroxyl position.
Usman et al., U.S. Pat. No. 5,804,683, describes a method for the removal of exocyclic protecting groups using alkylamines.
Wincott et al., U.S. Pat. No. 5,831,071, describes a method for the deprotection of RNA using ethylamine, propylamine, or butylamine.
Vinayak, U.S. Pat. No. 5,281,701, describes methods and reagents for the synthesis of RNA using 5′-O-protected-2′-O-alkylsilyl-adenosine phosphoramidite and 5′-O-protected-2′-O-alkylsilylguanosine phosphoramidite monomers which are deprotected using ethylthiotetrazole.
Usman and Cedergren, Trends in Biochem. Sci. 1992, 17, 334–339 describe the synthesis of RNA-DNA chimeras for use in studies of the role of 2′ hydroxyl groups.
Sproat et al., 1995 Nucleosides & Nucleotides 14, 255–273, describe the use of 5-ethylthio-1H-tetrazole as an activator to enhance the quality of oligonucleotide synthesis and product yield.
Gait et al., 1991, Oligonucleotides and Analogues, ed. F. Eckstein, Oxford University Press 25–48, describe general methods for the synthesis of RNA.
Koester and Coull, U.S. Pat. No. 4,923,901; Klem and Riley, U.S. Pat. No. 5,723,599; Furukawa et al., U.S. Pat. No. 5,674,856; Nelson, U.S. Pat. No. 5,141,813; Reed et al., U.S. Pat. No. 5,419,966; Caruthers and Matteucci, U.S. Pat. No. 4,458,066; Bhatt, U.S. Pat. No. 5,252,723; Weetall et al., 1974, Methods in Enzymology, 34, 59–72; Van Aerschot et al., 1988, Nucleosides and Nucleotides, 7, 75–90; Maskos and Southern, 1992, Nucleic Acids Research, 20, 1679–1684; Van Ness et al., 1991, Nucleic Acids Research, 19, 3345–3350; Katzhendler et al., 1989, Tetrahedron, 45, 2777–2792; Hovinen et al., 1994, Tetrahedron, 50, 7203–7218; Nippon Shinyaku, GB 2,169,605; Boehringer Mannheim, EP 325,970; Reddy and Michael, International PCT publication No. WO 94/01446; Akad. Wiss. DDR, E. German patent No. 280,968; and Bayer, W. German patent No. 4,306,839, all describe specific examples of solid supports for oligonucleotide synthesis and specific methods of use for certain oligonucleotides.
Zhang and Tang, International PCT Publication No. WO 97/42202; and Kitamura et al., 2000, Chem Lett., 10, 1134–1135 describe specific phosphitylating reagents and their use in oligonucleotide synthesis via in situ generation of 5′-O-protected nucleoside 3′-O-phosphoramidites.